OSTEOPONTIN ACTIVATION OF C-SRC IN HUMAN-MELANOMA CELLS REQUIRES THE CYTOPLASMIC DOMAIN OF THE INTEGRIN ALPHA(V)-SUBUNIT

In human melanoma cells, expression of the alpha(v) beta(3) integrin i s correlated with the metastatic potential. The expression of osteopon tin (OPN or OP), a protein ligand for the integrin alpha(v) beta(3), a lso correlates with metastatic potential of some tumors. Analysis of s ignal transduction, stimulated by OPN/alpha(v) beta(3) in human melano ma cells (M21), revealed activation of pp(60c-src) associated with the integrin. pp(60c-src) stimulation by OPN was dose dependent, and it w as inhibition in vitro by a tyrosine kinase inhibitor, herbimycin-A. T o determine the need for the cytoplasmic domain of the alpha(v)-subuni t, in the association of pp(60c-src) with alpha(v) beta(3), a cell lin e expressing truncated alpha(v) was studied. M21-L cell lacked alpha(v ) expression but stably transfected with complementary DNAs encoding a lpha(v) full length protein alpha(v)1018 or alpha(v)995 (lacking 23 ca rboxyl-terminal amino acids), and a fibroblast cell line (FG) expressi ng alpha(v) beta(5) but not alpha(v) beta(3), were used. Western analy sis and immune complex kinases assays of anti-alpha(v) immunoprecipita tes demonstrated that M21-L/alpha(v)995 cells did not exhibit pp(60c-s rc) association with alpha(v), whereas the alpha(v)1018 complementary DNA transfected cells and FG cells had pp(60c-src) associated with the alpha v integrins. Immunofluorescence analysis revealed pp(60c-src), alpha(v) beta(3) integrin, and actin distribution along the plasma mem brane of M21 cells. S-35-labeling of cells and analysis of complexes i mmunoprecipitated by a monoclonal antibody against alpha(v) beta(3) de monstrated association of actin with the immune complexes. We conclude that OPN stimulates pp(60c-src) kinase activity associated with the a lpha(v) beta(3) integrin and that the association requires the cytopla smic tail of the alpha(v) chain.